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1 Elucidating the diversity of microeukaryotes and epi-endophytes in the brown 2 algal holobiome 3 4

5 Marit F. Markussen Bjorbækmo1,2*, Juliet Brodie2, Ramiro Logares3, Stephanie 6 Attwood2, Stein Fredriksen4, Janina Fuss1, Kamran Shalchian-Tabrizi1, Anders Wold- 7 Dobbe1, Anders K. Krabberød1, David Bass2,5* 8 9 10 1. University of Oslo, Department of Biosciences, Section for Genetics and 11 Evolutionary Biology (EVOGENE), N-0316, Oslo, Norway

12 2. The Natural History Museum (NHM), Department of Life Sciences, London SW7 13 5BD, United Kingdom

14 3. Institut de Ciències del Mar (CSIC), Department of Marine Biology and 15 Oceanography, ES-08003, Barcelona, Catalonia, Spain

16 4. University of Oslo, Department of Biosciences, Section for Aquatic biology and 17 Toxicology (AQUA), N-0316, Oslo, Norway 18 19 5. Centre for Environment Fisheries and Aquaculture Science (CEFAS), Weymouth, 20 Dorset DT4 8UB, United Kingdom 21 22 23 * Correponding authors: 24 David Bass 25 e-mail: [email protected] 26 Phone: 27 28 Marit F. Markussen Bjorbækmo 29 e-mail: [email protected] 30 Phone:

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31 Abstract

32 Background

33 Brown algae (Phaeophyceae) are essential species in coastal ecosystems where they

34 form kelp forests and seaweed beds that support a wide diversity of marine life. Host-

35 associated microbial communities are an integral part of phaeophyte biology. The

36 bacterial microbial partners of phaeophytes have received far more attention than

37 microbial eukaryotes. The pre-requisite to understand the ecology of phaeophytes and

38 their host-associated microbes is to know their diversity, distribution and community

39 dynamics. To our knowledge, this is the first study to investigate phaeophyte-associated

40 eukaryotes (the eukaryome) using broadly targeting ‘pan-eukaryotic’ primers and high

41 throughput sequencing (HTS). Using this approach, we aimed to unveil the eukaryome

42 of seven large common phaeophyte species. We also aimed to assess whether these

43 macroalgae harbour novel eukaryotic diversity and to ascribe putative functional roles

44 to the host-associated eukaryome, based on taxonomic affiliation and phylogenetic

45 placement.

46

47 Results

48 Our sequence dataset was dominated by phaeophyte reads, from the host species and

49 potential symbionts. We also detected a broad taxonomic diversity of eukaryotes in the

50 phaeophyte holobiomes, with OTUs taxonomically assigned to ten of the eukaryotic

51 major Kingdoms or supergroups. A total of 265 microeukaryotic and epi-endophytic

52 operational taxonomic units (OTUs) were defined, using 97% similarity cut off during

53 clustering, and were dominated by OTUs assigned to Stramenopiles, Alveolata and

54 Fungi. C Almost one third of the OTUs we detected have not been found in previous

55 molecular environmental surveys, and represented potential novel eukaryotic diversity.

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56 This potential novel diversity was particularly diverse in phylogenetic groups

57 comprising heterotrophic and parasitic organisms, such as labyrinthulids and

58 Oomycetes, Cercozoa, and Amoebozoa.

59

60 Conclusions

61 Our findings provide important baseline data for future studies of seaweed-associated

62 microorganisms, and demonstrate that microeukaryotes and epi-endophytic eukaryotes

63 should be considered as an integral part of phaeophyte holobionts. The potential novel

64 eukaryotic diversity we found and the fact that the vast majority of macroalgae in

65 marine habitats remain unexplored, demonstrates that brown algae and other seaweeds

66 are potentially rich sources for a large and hidden diversity of novel microeukaryotes

67 and epi-endophytes.

68

69

70

71 Keywords [3-10]

72 Symbiome, epiphyte, endophyte, protist, microeukaryote, eukaryome, Phaeophyceae,

73 seaweed, holobiont, fungi

74

75

76

77 Background

78 Seaweeds are essential to the health of our planet, providing core ecosystem services,

79 and food and shelter to a wide diversity of marine life, from microbes to mammals, [1,

80 2 and references therein]. Brown algal seaweeds (Phaeophyceae) are Stramenopiles, a

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81 radiation of eukaryotes which is phylogenetically distant from red- and green algal

82 seaweeds (Archaeplastida; Chlorophyta and Rhodophyta). The phaeophytes are one of

83 the most diversified groups of benthic algae and comprise approximately 2000 species,

84 encompassing complex multicellular species which have diversified and evolved since

85 the Mesozoic Era ~250 MYA [2, 3]. Large phaeophytes, especially those in the orders

86 Laminariales, Fucales and Tilopteridales are dominant members of intertidal and

87 shallow subtidal ecosystems worldwide, where they function as ecosystem engineers

88 forming complex underwater forests. These coastal ecosystems are some of the most

89 productive habitats on the planet [4], where seaweed beds and kelp forests are

90 indispensable biodiversity hotspots, which also provide habitat and nursery for a wide

91 range of taxa, including many commercially important species [5]. Phaeophytes have

92 been shown to accommodate up to 100.000 individuals of different invertebrate species

93 per square metre [6].

94 Phaeophytes and other seaweeds also harbour a wide diversity of microbial

95 epibionts and endobionts, comprising eukaryotes [7-9], prokaryotes [10-12] and viruses

96 [13-16]. These, together with the host, are collectively referred to as the seaweed

97 holobiont and can be considered as a localized ecosystem living on and in a host [10,

98 17-19]. It is increasingly recognized that host-associated microbial communities

99 (microbiomes) are an integral part of the host biology, exerting diverse and strong

100 influences on their hosts [19, 20]. While the term microbiome usually refers mostly or

101 exclusively to bacteria, it is important to consider the whole microbial symbiome,

102 including microbial eukaryotes (microeukaryotes) and larger epiphytic, epizootic, and

103 endophytic symbionts, to enable a comprehensive understanding of holobiont

104 functioning [19, 21].

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105 Symbiotic interactions in its broadest sense refers to all intimate ecological

106 interactions between two species [22]. According to the fitness effect on the members

107 involved in the symbiotic relationship, the associations cover a wide spectrum of

108 interactions along the gradual continuum between positive mutualism and negative

109 parasitism. Some symbiotic interactions are obligate while other are more temporary

110 and fluctuating, and the nature of the interaction can vary depending on the symbiont’s

111 interactions with its hosts, other symbionts and environmental conditions [20]. To date,

112 bacterial symbionts of phaeophytes have received most study [e.g., 10, 11, 12, 23], and

113 have been shown to represent complex and highly dynamic relationships that can have

114 everything from fundamental to detrimental effects on their hosts [10, 24-28].

115 Microeukaryotes in the phaeophyte symbiome are far less well known. Only a

116 small number of studies have investigated these associations, mostly using traditional

117 culturing/cell isolation methods or targeted molecular approaches. These studies

118 demonstrate that a broad taxonomic diversity of microeukaryotes are associated with

119 phaeophytes, including surface dwelling heterotrophic diatoms, dinoflagellates and

120 ciliates [8], ‘naked amoeba’ [29], epiphytic and endophytic diatoms [30-32], and green

121 algal endophytes [33], in addition to parasitic or saprotrophic labyrinthulids [34, 35],

122 oomycetes [7, 36, 37], phytomyxids [38-40] and fungi [9, 41-44]. The nature of these

123 microeukaryote-host relationships is mostly unknown, although some symbionts can

124 have detrimental effects on their macroalgal hosts, for example phytomyxids [38-40],

125 oomycetes [7, 36, 37] and chytridiomycete fungi [41]. Other microeukaryotes are

126 suspected to have a beneficial effect on their hosts, for example fungi that are mutualists

127 [9, 45, 46] and fungal endophytes that might protect seaweeds against pathogenic

128 protists [44].

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129 Adding to the complexity of the microeukaryotic biodiversity associated with

130 phaeophytes, their thalli are often overgrown with a wide variety of smaller seaweeds

131 which represents a continuum between epiphytism and endophytism. These epi-

132 endophytes can have negative effects such as imposing physical and physiological

133 stress on their host, or positive ecosystem effects such as increased available habitats

134 and food for both macroscopic and microscopic life [47]. As macroalgal symbiomes

135 comprise multi-cellular algae as epiphytes [48, 49] and potentially pathogenic

136 endophytes [50-53] in addition to microeukaryotes, the almost complete lack of

137 knowledge of these eukaryotes in a holobiont context/perspective is a fundamental

138 knowledge gap which needs to be filled in order to improve our understanding of

139 phaeophyte holobionts.

140 The overall aim of this study was to unveil the eukaryome of seven large

141 phaeophytes which are key components in coastal ecosystems: Fucus vesiculosus, F.

142 serratus, Himanthalia elongata and Ascophyllum nodosum (order Fucales), Laminaria

143 digitata and Saccharina latissima (order Laminariales) and Saccorhiza polyschides

144 (order Tilopteridales). We achieved this aim by using using broadly-targeted pan-

145 eukaryotic primers and High Throughput Sequencing (HTS). This application in host

146 environments has so far been limited due to the technical limitation of co-amplifying

147 host DNA when using ‘universal’ eukaryotic primers [20]. However, with sufficient

148 sequencing depth now tractable to amplify host-associated eukaryotes much more

149 comprehensively than was previously possible, this will become increasingly

150 established as a standard tool for investigating host-associated eukaryomes [54, 55].

151 We also aimed to assess whether phaeophytes harbour a novel or differentially

152 structured diversity of (micro)eukaryotes, and to ascribe putative functional roles to the

153 microeukaryotes associated with these phaeophytes (e.g., putative parasites).

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154

155 Results

156 Composition of the brown algal eukaryome

157 A total of 256 eukaryotic Operational Taxonomic Units (OTUs) were defined by

158 UPARSE and post-UPARSE trimming. Their read abundance in each library are shown

159 in Supplementary Table S1. Each library represented five phaeophyte samples pooled

160 according to species and geographic location, and is hereafter referred to as samples.

161 The richness recovered from each sample ranged from 26 to 101 OTUs, and the

162 taxonomic profile of these are shown as proportional OTU abundance in Fig. 1,

163 Supplementary Table S1. The sequence dataset was dominated by phaeophyte reads

164 (97.3% of all sequences, Supplementary Table S2). Phaeophytes also displayed the

165 highest OTU richness (18.9% of the total OTUs; Fig. 2, and accountable for 27.3-

166 70.4% of the OTUs per sample; Supplementary Table S2). Nevertheless, there was a

167 broad taxonomic diversity of microeukaryotes in the phaeophyte samples with OTUs

168 taxonomically assigned to ten of the eukaryotic major Kingdoms or supergroups (Fig.

169 1, Fig. 2).

170

171 Phylogenetic diversity of eukaryotic OTUs

172 Phylogenetic analyses were conducted for taxonomic groups displaying either high

173 taxonomic diversity and/or taxonomic groups previously documented to be in a

174 symbiotic relationship (sensu lato) with phaeophytes. The resulting phylogenies and

175 heat maps of proportional read abundance (log10) of each OTU in the different samples

176 are shown in Figs. 3-7 and Supplementary Figs 1-4, which include OTUs and the closest

177 sequence matches in NCBI GenBank as of 05.05.2020 (re-blasted 11.04.2021).

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178 For some taxonomic groups, the heatmaps indicated that the majority of OTUs

179 were only detected associated with one phaeophyte sample, such as Cercozoa (Fig. 4),

180 Fungi (Fig. 5), Ciliophora (Supplementary Fig. 1), Apicomplexa and Perkinsea

181 (Supplementary Fig. 2), Dinoflagellata (Supplementary Fig. 4) and Amoebozoa and

182 Centroheliozoa (Supplementary Fig. 3). However, most of these taxonomic groups also

183 comprised OTUs that were found in multiple samples, across host orders and

184 geographic locations (e.g., the fungal ascomycete OTU 226; Fig. 5, the ciliate OTU 89;

185 Supplementary Fig. 1, and the centroheliozoan OTU 13; Supplementary Fig. 3 were

186 found in samples belonging to Fucales, Tilopteridales and Laminariales from both

187 Norway and the UK). For other taxonomic groups such as Bacillariophyta (Fig. 6) and

188 Labyrinthulomycetes & Oomycetes (Fig. 3), the heatmaps show that a large proportion

189 of the OTUs were detected in multiple samples.

190 Several OTUs clustered with reference sequences that have been found

191 associated with various hosts in previous studies, such as the labyrinthulid and

192 oomycete OTUs clustering with Aplanochytrium (Labyrinthlida) and Olpidiopsidales

193 (Fig. 3), and the cercozoan vampyrellid OTUs clustering with a reference sequence

194 previously detected in samples of brown alga (Fig. 4).

195

196 Novel diversity in the brown algal eukaryome

197 We found that ~30% of the OTUs had low percentage identity (<95 %) with any known

198 close relatives in reference sequence databases and represent potential novel taxonomic

199 diversity (Supplementary Table 1). The majority of OTUs taxonomically and

200 phylogenetically placed within Fungi, Bacillariophyta and ciliates displayed high

201 percentage identity (>95 %) to known reference sequences (Fig. 5; Fig. 6; and

202 Supplementary Fig. 1, respectively). Conversely, within Labyrinthulomycetes and

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203 Oomycetes (Fig. 3), Cercozoa (Fig. 4) and Amoebozoa and Centroheliozoa

204 (Supplementary Fig. 3), about 50% of the OTUs had low similarity to known reference

205 sequences (<95 %) which indicated that these OTUs represented potential novel

206 diversity. Some of this potential novel diversity was represented by specific clades in

207 the phylogenetic trees, such as a well-supported labyrinthulid clade within

208 Thraustochytrida that encompassed four OTUs with very low percentage identity

209 (ranging between 84.1% to 89.6%). Similarly, within Cercozoa, eight of the nine

210 Vampyrellida, Phytomyxea, and Novel Clade 9 OTUs had low similarity to reference

211 sequences (from 85.2% to 93%; Fig 4).

212

213 Green (Chlorophyta), red (Rhodophyta) and Brown (Phaeophyceae) algae:

214 In addition to protist taxa, we also detected OTUs from green, red, and (non-host-

215 derived) brown algae (Fig. 7). The majority of the nine green algal OTUs (Fig. 7) were

216 very similar (>99% identity) to characterized and sequenced Ulvales taxa such as

217 Ulvella (formerly Acrochaete) leptochaete and Umbraulva japonica. Only a single

218 green alga (OTU 264) displayed low similarity (90.4%) to reference sequences, and

219 clustered with Lithotrichon pulchrum, Ulvales (Fig. 7). Similarly, the five red algal

220 OTUs (Fig. 7) displayed >98% similarity to taxa including Ceramium sp. and

221 Cryptopleura ramosa.

222 There were in total 50 phaeophyte OTUs. Significant levels of microdiversity

223 in the V4 OTUs were seen in the phaeophyte clade. OTUs corresponding to the host

224 species were inferred on the basis of their phylogenetic proximity to, and high

225 proportions of reads associating with, reference sequences from that host, either from

226 GenBank or sequences generated in this study. Host OTUs are indicated by labeled

227 brackets on Fig. 7. Many of the OTUs derived from the host species were present in

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228 samples from multiple host species, albeit generally represented with lower read

229 abundance in the ‘non-host’ samples.

230 However, other OTUs did not cluster with host-derived sequences, or from the

231 longer 18S sequences generated by cloning amplicons from the host. The 26 OTUs in

232 the “uncharacterized clade” on Fig. 7 were clearly distinct from any characterized or

233 environmental sequences in reference sequence databases. This clade encompassed

234 OTUs that were mainly detected in the Fucus sp. samples, but six of them were also

235 detected in lower abundance in other phaeophyte samples (Fig. 7). Additionally, seven

236 OTUs in the A. nodosum clade may be host sequences but are obviously different in

237 sequence from the cloned sequences generated from that host, and OTUs 2 and 326,

238 presumably deriving from that host (Fig. 7).

239 Some OTUs grouped strongly with known epiphytic lineages, such as the three

240 OTUs clustering with genera Pylaiella and Halothrix. These OTUs were found in all,

241 or in the majority, of the different pheaophyte hosts (Fig. 7). In the same clades were

242 the brown algal endophytes isolated and sequenced by Attwood (2005), published here

243 for the first time, and represented on Fig. 7, all in the ‘brown endophytes’ clade. These

244 endophytes were identified as Myrionema strangulans, isolated from Fucus serratus,

245 Mastocarpus stellatus, Chorda filum, Osmundea pinnatifida, and Chondrus crispus;

246 Microspongium tenuisimum, isolated from Osmundea osmunda and C. crispus;

247 Streblonema sp. isolated from M. stellatus; Mytriotrichia clavaeformis and Ectocarpus

248 sp. from C. crispus; and Chorda filum from a larger C. filum individual. Light

249 microscopy images of the isolated endophytes are shown in Fig. 8.

250 Three of our OTU sequences occurred in the same clade as these endophytes:

251 OTU 9 and some 18S clone sequences from S. polyschides clustering with Pylaiella,

252 and OTUs 70 and 146, clustering most closely with Myriotrichia and Myrionema (Fig.

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253 7). All of these were strongly associated with F. vesiculosus and S. polyschides

254 individuals, but they were present in almost all host samples.

255

256 Analyses of community composition

257 The clustering of samples was consistent irrespective of whether host OTUs were

258 included in the analyses (Fig 9: A-B). The three F. vesiculosus samples clustered with

259 F. serratus and S. polyschides in both plots (Fig 9: A-B), which indicated that these

260 have a similar OTU composition. The A. nodosum sample showed less similarity with

261 Fucus and S. polyschides when the phaeophyte OTUs were excluded from the analysis

262 (Fig. 9, A-B). Himanthalia elongata did not cluster with any other phaeophyte hosts

263 and appeared highly dissimilar from the others both including and excluding

264 phaeophyte OTUs (Fig. 9; A-B). Analyses of similarity (ANOSIM) was highly

265 significant both with and without phaeophyte OTUs included (p-value 7e-04 and 0.03

266 respectively), which supported the dissimilarity observed between the phaeophyte host

267 genera in the NMDS plots (Fig. 9: A-B).

268 Dissimilarity between host genera (SIMPER values) ranged from 31.4%

269 (Laminaria spp. vs Saccharina spp.) to 99.9% (Ascophyllum spp. vs Himanthalia spp.)

270 in the dataset including phaeophyte OTUs (Table 1, Supplementary Table S3), where

271 certain host derived OTUs (OTU 1, 2, 3 and 5) were the main contributors to the

272 observed dissimilarity. These results also supported some of the OTUs inferred to

273 derive from the host species based on their phylogenetic placement and read abundance

274 (Table 1, Fig. 7). When phaeophyte OTUs were excluded the host genera were all

275 highly dissimilar, with SIMPER values ranging from 83.3% (Ascophyllum spp. vs

276 Saccorhiza spp.) to 99.4% (Ascophyllum spp. vs Himanthalia spp.). The main

277 contributors to the observed dissimilarity between the phaeophyte host genera were

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278 green algal, red algal, fungal and oomycete OTUs in addition to an unassigned OTU

279 (Table 2). There were also several OTUs assigned to a wide range of taxonomic groups

280 that had some contribution to the dissimilarity (Supplementary Table S3).

281 IndVal analyses of the dataset including phaeophyte OTUs, indicated that six

282 OTUs were preferentially associated with Ascophyllum (Table 3), of which five were

283 taxonomically assigned to Phaeophyceae. Four of these OTUs clustered within the

284 clade labelled “A. nodosum” in the phylogenetic tree (Fig. 7, Table 3), while the fifth

285 OTU was placed in the “Uncharacterised” clade, represented with reads in all

286 phaeophyte libraries (Fig. 7). The final OTU (25) was a fungal OTU found in all the

287 libraries except L. digitata (UK), but with higher read abundance in Ascophyllum than

288 in the other host genera (Fig. 5). OTU 25 was also the only one found to be significant

289 by the IndVal analysis of the dataset excluding phaeophytes (p-value 0.0446; Table 2).

290

291 Shared OTUs

292 Although our analyses suggested a large degree of dissimilarity between the different

293 phaeophyte hosts (Table 1, 2), 70 OTUs were found in multiple samples across the host

294 orders Fucales, Laminariales and Tilopteridales (Fig. 10, Table 4). Of these, 25 OTUs

295 were detected in samples belonging to all of the three host orders, of which 15 belonged

296 to Phaeophyceae, including the three brown algal endophyte OTUs (Fig. 7), three OTUs

297 from the “uncharacterized” clade (Fig. 7) and several host OTUs that were found in

298 high abundances in the respective host samples, but also present in lower abundance in

299 several of the other phaeophyte samples. The shared OTUs also included five diatoms

300 which clustered with known epiphytes and endophytes of macroalgae in the

301 phylogenetic tree (Fig. 6, Table 4).

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302 Twenty OTUs were found in samples belonging to Fucales and Laminariales

303 (Table 4), including Fungi, Oomycetes and Labyrithulomycetes (Table 4, Fig. 5, 3).

304 The 22 OTUs that overlapped between Fucales and Tilopteridales comprised several

305 diatom and green algal OTUs (Table 4, Fig 6, 7), and two Oomycete OTUs that

306 clustered within Olpidiopsidales (Table 4, Fig. 3). The OTUs that were unique to

307 Fucales, Tilopteridales and Laminariales are shown in Supplementary Table S4.

308

309 Discussion

310 Broad diversity of microeukaryotes associated with brown algae

311 To our knowledge, this is the first study that has investigated the diversity of eukaryotes

312 associated with brown algae using high throughput sequencing. Our findings show that,

313 although the sequence data was dominated by brown algal reads, we were able to

314 identify a broad diversity of microeukaryotes representing most of the main branches

315 in the eukaryotic tree of life (Fig 1, 2).

316 Some of the brown algae we investigated in this study displayed a higher

317 diversity of microeukaryotes compared to others. Particularly, we detected a higher

318 diversity associated with Fucus vesiculosus, F. serratus (Fucales) and Saccorhiza

319 polyschides (Tilopteridales) than Laminaria digitata and Saccharina latissima

320 (Laminariales). There are several factors/variables which may have affected the

321 observed diversity and community composition. Density and composition of

322 microorganisms associated with brown algae have been shown to vary at several scales:

323 e.g., among host species, between individuals of the same host species, different thallus

324 areas of individual hosts, the kind of surface tissue available for microepiphyte

325 colonization, different habitats and location in the intertidal zone, seasons, different

326 stages of the life cycle, and different longevity of the host [e.g., 19, 25, 32, 56, 57-59].

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327 In addition, the interaction between the settlers affects the formation of specific

328 communities [47, 56], including chemical interactions and signalling between the

329 microbial symbionts [44, 60, 61]. Seaweeds themselves also, to various degrees have

330 physical and chemical antifouling defence mechanisms which prevents settlement of

331 microorganisms and epiphytes [31, 62, 63]. For this study, we may also have randomly

332 sampled different microhabitats of the brown algal thalli, resulting in a highly variable

333 diversity of microeukaryotes.

334 The community composition of microeukaryotes associated with Himanthalia

335 elongata stood out as very dissimilar to the other phaeophytes (Fig. 9 B). This seaweed

336 differs from the other phaeophytes in the type of life cycle and the tissue type sampled

337 for this study. H. elongata is biannual, but the elongated thongs which accounts for

338 ~98% of the thallus is reproductive tissue that degrades at the end of the season

339 (reproductive season from late July to ~January), leaving only the vegetative basal disc

340 [64]. The tissue type sampled from H. elongata were therefore different and most likely

341 younger than the tissues sampled from the other phaeophytes, which could explain the

342 distinctive and low diversity of microeukaryotes detected in this host. A recent study

343 also reported low diversity of bacteria associated with H. elongata [65].

344

345 Eukaryotic epiphytes and endophytes in the seaweed holobiont:

346 Seaweed surfaces provide nutrient-rich and important habitats for epiphytic and

347 endophytic organisms, from unicellular microbes such as bacteria and protists to

348 multicellular, usually filamentous seaweeds [e.g., 10, 12, 47, 56, 66]. These

349 associations can be either facultative (i.e. the epi-endophytes can grow on a variety of

350 biotic and/or abiotic substrata) or obligate (dependent on one or more specific hosts).

351 Like many biological categorizations, the boundaries between epiphytes and

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352 endophytes in real-life can be blurred; the nature of the association can be affected by

353 both biotic and abiotic factors, and can range from mutualism to parasitism [47, 67,

354 68].

355

356 Microeukaryotic epi-endophytes:

357 Diatoms are regularly found in the biofilm on seaweed surfaces, where they live as

358 epiphytes, and often occur in large numbers [32, 69-71]. Although many diatoms

359 associated with phaeophytes are epiphytes, there are also studies which have

360 demonstrated that some diatom species, such as the ones belonging to the genera

361 Navicula and Cocconeis can live as endophytes, intracellularly in tissues of both brown

362 algae and red algae [30, 72-74]. Mayombo et al. [75, 76] recently suggested that some

363 diatom taxa might have adapted to an endophytic life to avoid the antifouling

364 mechanisms of their hosts. Several of the OTUs from our study which were

365 taxonomically and phylogenetically assigned to different species of Naviculales,

366 Cocconeidales and Fragilariales (Fig. 6), were present in multiple samples/hosts

367 independent of geographic location. Our molecular data in combination with previous

368 observational studies [30, 32, 72-76] supports the hypothesis that some diatom species

369 such as Navicula sp. and Cocconeis sp., might have a more intimate, potentially

370 endophytic, association with seaweeds. This, however, remains to be tested in future

371 studies.

372 Ciliates represent another ubiquitous group of microeukaryotes living as

373 epiphytes in the biofilm on seaweeds [8, 77], and was also the dominating group of

374 alveolates in our study (Fig. 2). Many ciliates are predators/grazers feeding on bacteria

375 and microeukaryotes [8], whereas other ciliates can be involved in symbiotic

376 interactions [78]. Most of the ciliates detected in our study were only found in single

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377 samples (Supplementary Fig. 1), which might indicate that these ciliates were “random

378 visitors”. A few of the OTUs were, however found in several samples from both

379 Norway and the UK, and were highly similar to reference sequences from different

380 marine environmental samples, which suggest that these ciliates are generalist biofilm

381 formers on different organisms and substrates. However, one of these OTUs (OTU 256,

382 Spirotrichea, Supplementary Fig. 1) was almost identical to a reference sequence found

383 in samples of Ascophyllum nodosum used as live bait wrapping [79], which could point

384 towards that some of these ciliates have a closer association with brown algae. As

385 emphasised by del Campo et al. [20], “it is likely that many microeukaryotes routinely

386 detected or observed/cultured from environmental samples may also be host associated,

387 even if transiently”.

388

389 Host-associated phaeophytes, chlorophytes and rhodophytes

390 In addition to the phaeophyte OTUs clearly deriving from the host species, we detected

391 a diversity of other sequence types from putative brown algal epi-endophytes (Fig. 7).

392 The diversity of ‘OTUs’ apparently deriving from the host organisms was much higher

393 than expected. This may partly be caused by artefactual sequence variants amplified to

394 a discernible extent by the depth of Illumina sequencing. But the distinctiveness of

395 some of these OTUs from obviously host-derived sequences was so great in some cases

396 (e.g., the strongly Fucus-associated ‘uncharacterised’ clade in Fig. 7) that we propose

397 that these represented hitherto unknown epi-endobionts. In other cases we have

398 conservatively ascribed OTU diversity clustering with host sequences (e.g., the clades

399 bracketed as A. nodosum and H. elongata in Fig. 7) as being host-derived, but there

400 remains the possibility that they represented different organisms.

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401 Some of the phaeophyte OTUs detected in our study, such as the ones clustering

402 with epiphytic Pylaiella and Halothrix and the other phaeophyte epi-endophytes (Fig.

403 7), clearly also represented epi-/endophytic lineages [e.g., 48, 58, 80]. These OTUs

404 were detected in all the phaeophyte hosts included in this study, indicating a broad

405 distribution and host range.

406 By analysing sequences from cultured phaeophyte symbionts of brown algae

407 and red algae (Attwood 2005) we show that the epi-endophyte genera Microspongium,

408 Myriotrichia, Myrionema, and Streblonema form a clade with Chorda and Ectocarpus

409 (growing as epi-endophytes), and previously sequenced phaeophyte epi-endophytes

410 Pylaiella, Halothrix, and Scytosiphon, which all together form a maximally supported

411 clade with Saccorhiza. While this significantly expands the 18S sequence

412 representation of phaeophyte epi-endophytes, it does not account for the vast majority

413 of host-associated phaeophyte sequences in our data that are not very obviously host-

414 derived. Further culturing and is situ experiments are required to explain these

415 suggestive findings, which might represent microscopic life-stages (zoospores,

416 gametophytes, gametes and juvenile sporophytes) of brown algae growing epi-

417 /endophytically on substrata, including on macroscopic sporophytes of other seaweeds

418 [81-84]. This can also explain why the host-derived OTUs were detected in lower

419 abundance in the other brown algal samples (Fig. 7).

420 Another possible explanation is that small filamentous brown algae were

421 growing epi-endophytically in the larger brown algal hosts [48, 85]. Such endophytic

422 infections are described as common diseases of brown algae [e.g., 52, 53, 67, 68, 86].

423 Hosts containing brown algal endophytes can either be asymptomatic, show weak

424 symptom such as dark spots or warts, or present severe symptoms such as

425 morphological changes to the brown algal host tissues [52, 67]. Substrata and brown

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426 algal thalli often contain many more brown algal endophytes than expected based on

427 visual inspections prior to culturing [47, 53, 85].

428 The green algal OTUs were all similar to reference sequences of taxa that are

429 known to be endophytes in red (Chondrus crispus Mastocarpus stellatus, Osmundea

430 spp. and Dumontia contorta) and brown algae (Fucus serratus and Chorda filum) [33,

431 87, 88] .

432 All red algal OTUs were similar to reference sequences of taxa that are common

433 epiphytes on seaweeds and a variety of other substrata. Several of the taxonomic names

434 for the reference sequences included in the phylogeny are out of date (Fig. 7):

435 Ceramium spp. is an aggregate of species (i.e. Ceramium rubrum in Fig. 7) which are

436 members of the order Ceramiales together with Calithamnion spp., Polysiphonia sensu

437 lato (Polysiphonia fucoides is now Vertebrata fucoides) and Cryptoleura spp. Red algal

438 endophytes are common in other rhodophytes and chlorophytes (REF!). We did not,

439 however find any endophytic rhodophytes in our study, which might indicate that

440 phaeophytes are not prone to endophytic infections by rhodophytes, but instead are

441 more susceptible to endophyte infestations by chlorophytes and endophytic

442 phaeophytes.

443

444 Novel diversity and putative parasites

445 We demonstrate that brown algal holobionts encompass potential novel eukaryotic

446 diversity, as almost one third of the OTUs we detected have not been found in molecular

447 environmental surveys before. This was more pronounced in some phylogenetic groups

448 than in others, in particular the Labyrinthulids and Oomycetes (Fig. 3), Cercozoa (Fig.

449 4), Amoebozoa and Centroheliozoa (Supplementary Fig. 3). Some of the OTUs were

450 taxonomically and phylogenetically placed with known and putative parasites of

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451 seaweeds. Our findings and the fact that the vast majority of macroalgae in marine

452 habitats remain unexplored, demonstrates that brown algae and other seaweeds are

453 potentially rich sources for a large and hidden diversity of novel microeukaryotes.

454 Labyrinthulomycetes are heterotrophic Stramenopiles which are abundant and

455 diverse in a wide range of marine and freshwater habitats where they play important

456 roles as saprotrophs/decomposers [89, 90]. Some Labyrinthulomycetes are also known

457 as important symbionts (parasites, mutualists or commensals) of marine organisms,

458 including brown algal seaweeds [91-93]. Although most thraustochytrids are free

459 living, a few species have been associated with disease in marine metazoans, including

460 the quahog parasite QPX which parasitizes clams. Several of the OTUs in our study

461 clustered in three highly supported clades within Labyrinthulida (Fig 3), and had high

462 similarity to Aplanochytrium labyrinthulids associated with various hosts such as corals

463 [94], the pseudoparenchymatous brown alga Elachista sp. [93], seagrass [95] and sea

464 stars [96].

465 Certain Oomycetes are common parasites of brown and red algae [37, 97-99],

466 and Olpidiopsis species have been shown to have cosmopolitan occurrence and broad

467 host ranges [98, 100]. In this study, we found OTUs clustering within Olpidiopsidales.

468 Some of these were highly similar to reference sequences of Anisolpidium rosenvingei

469 and A. ectocarpii (Fig 3), which are parasites of filamentous brown algae [36]. Others

470 displayed high similarity to Pontisma lagenidioides and “Ectrogella” (lnfLicSC1,

471 SC2-a, SC3 and SC4) which parasitizes the diatom Licmophora sp. and the red algae

472 Ceramium virgatum [101, 102]. Further, we detected two novel OTUs with no close

473 relatives among available reference sequences (OTU 214; 90.9 % identity to JN635125

474 and OTU 290; 92.4 % identity to KT273921, Fig. 3), of which the closest known

475 relative is an Oomycete parasitizing diatoms [101, 103].

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476 Cercozoan diversity was dominated by vampyrellid amoebae, with four OTUs

477 forming a distinct clade with GU385680, which was previously detected on

478 Ascophyllum nodosum used as live bait wrapping [79]. Vampyrellids exhibit a wide

479 diversity of feeding strategies and often feed omnivorously; it is striking that this clade

480 comprises only lineages associated with brown algae, suggesting a specific association.

481 Phytomyxids, biotrophic parasites of angiosperms, oomycetes, and stramenopile algae

482 [39], were represented by two OTUs in a clade of marine sediment-derived sequences,

483 so there is no direct evidence for a specific interaction with macroalgae. OTU 242

484 groups with endomyxan Novel Clade 9 [104], members of which are found in a wide

485 range of environment types and have been hypothesized to be parasites of algae.

486 Fungi associated with brown algae can have everything from detrimental to

487 beneficial effects on their hosts [105, and references therein]. Two of the Ascomycota

488 OTUs clustered with taxa known to be parasites of brown algae such as Sarocladium

489 and Acremonium [106] (Fig. 5). Several of the basidiomycete OTUs clustered with

490 reference sequences of Cryptococcus and Cystofilobasidium that have previously been

491 found associated to various marine invertebrates, algae and seaweeds (Fig. 5) [107-

492 110]. In addition, within Chytridiomycota there were several OTUs that clustered with

493 Rhizophydium sp., which comprise parasites with broad host ranges known to infect

494 both macroalgae and protists [111, 112]. One OTU (OTU 76; Fig. 5) also displayed

495 high similarity to Chytridium polysiphoniae, a parasite of brown algae [113, 114]. The

496 final chytrid OTU clustered with Mesochytrium penetrans, a parasite of the unicellular

497 green algae Chlorococcum minutum [115], indicating that this could represent a parasite

498 of one of the green algae found in our study, or that relatives of M. penetrans have a

499 broader host range than currently known.

500

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501 Conclusions

502 Brown algal holobionts are major marine habitat formers that provide food and shelter

503 for diverse communities of both macro- and microorganisms. Our findings demonstrate

504 that microeukaryotes should be considered as an integral part of brown algal holobionts,

505 and provides important baseline data for future studies of seaweed-associated

506 microorganisms. The prerequisite to understand the ecology of brown algae and their

507 host-associated microbes, is to know their diversity, distribution and community

508 dynamics. Consequently, in order to obtain an understanding of the interdependence of

509 all the different players in brown algal holobionts it is critical to include the so far

510 largely neglected eukaryotic and microeukaryotic partners in future holobiont

511 investigation. It is however also important to remember that microeukaryotes interact

512 not only with the host but also with the prokaryotes and viruses that share the same host

513 environment, and future holobiont surveys should ideally include all of these

514 components.

515

516 Methods

517 Sample collection

518 Five individuals each of Ascophyllum nodosum, Fucus vesiculosus, Laminaria digitata

519 and Saccharina latissima were sampled from the Oslofjord (59°40¢26.9²N,

520 10°35¢13.199²E), while five individuals each of F. vesiculosus, F. serratus

521 Himanthalia elongata, Saccorhiza polyschides and L. digitata were collected at

522 Newton’s Cove (Dorset, UK: 50°36¢18²N, 2°26¢58²W) in October 2015. Samples were

523 collected by free diving and shore-based collection, kept cool in separate containers of

524 seawater while transported to molecular laboratories where they were immediately

525 subjected to subsampling of tissues for molecular analyses.

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526 All samples were handled under laminar flow hoods to limit their exposure to

527 airborne contaminants. The brown algae were rinsed in sterile artificial seawater (ASW)

528 to remove loosely attached organisms (but not true epibionts) and debris from the

529 surface. This was done in three consecutive steps by vortexing the samples in sterile 50

530 mL tubes with ASW for 5-10 seconds. The rinsed individuals were placed in sterile

531 petri dishes, and one subsample of the algal thallus (approximately 1 cm2 squares) per

532 individual was excised and placed in separate 2 mL tubes. All algae looked healthy; we

533 did not specifically target potentially infected tissues.

534 The samples were freeze-dried under sterile conditions by perforating the

535 sample tube lids and placing the sample tubes inside sterile culture flasks with 0.2 um

536 filter caps (Thermo ScientificTM NunclonTM). After freeze-drying, samples were

537 weighted, placed in new 2 mL sample tubes, and stored at -80 °C until DNA extraction.

538

539 Endophyte sampling

540 As part of a previous student project (Attwood 2005), brown algal endophytes were

541 selected from the B. Rinkel collection at The Natural History Museum, London. The

542 samples were isolated from hosts as shown in Table 5. Partial 18S-ITS1 rDNA

543 sequences were amplified using the phaeophyte-specific primer DICI [116] and the

544 general primer TW7 [117]. The 18S region of this amplicon did not overlap with the

545 V4 region sequenced by the metabarcoding approach, so longer 18S amplicons were

546 generated from the brown macroalgal samples used for this study as described below,

547 to enable as much direct comparison of our newly generated sequences with those from

548 the endophytes. Full details of the DNA extraction, PCR, and sequencing methods used

549 for the endophytes are given in Attwood 2005.

550

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551 Molecular methods

552 The freeze-dried samples were mechanically disrupted by adding two sterile tungsten

553 carbide beads to each sample tube and tissue-lyzed at 20Hz for 2 minutes, or longer,

554 until the tissues were completely pulverised. Thereafter, a modified lysis buffer [118]

555 was added to the tubes proportional to sample weight. This lysis buffer contains

556 antioxidant compounds such as PVP (Polyvinylpyrrolidone) and BSA (Bovine Serum

557 Albumin) that bind polyphenols, and has high salt concentration which decrease the

558 levels of coextracted polysaccharides. The samples were homogenised and divided in

559 two or more tubes so that the sample used for DNA isolation did not exceed 20 mg dry

560 weight tissue. DNA was extracted following the protocol by Snirc et al. [118].

561 PCR amplification of the V4 region of the 18S rDNA gene was performed with

562 ‘pan eukaryotic’ primers; the forward primer used was a slightly modified version of

563 the TAReuk454FWD1 primer (5’-CCAGCASCYGCGGTAATWCC-3’) and reverse

564 primer TAReukREV3 (5’-ACTTTCGTTCTTGATYRA-3’) [119]. PCR reactions (25

565 μl) contained 1x KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 0.4 μM of each

566 primer, 0.5 mg/mL Bovine serum albumin (BSA; Promega) and 1 μl genomic DNA.

567 The PCR program had an initial denaturation step at 98°C for 2 min, 15 cycles of 30 s

568 at 98°C, 30 s at 53°C and 45 s at 72°C, then 20 similar cycles except that the annealing

569 temperature was 48°C, and a final elongation step at 72°C for 10 min. DNA was titrated

570 into several dilutions to take into account potential inhibiting substances from the

571 brown algae. Each brown algal individual sample was amplified separately. PCR

572 products were purified and eluted (20 μl) with ChargeSwitch PCR Clean-Up kit

573 (ThermoFisher Scientific), and quantified with the dsDNA BR Assay Kit and Qubit 2.0

574 Fluorometer (ThermoFisher Scientific), before they were pooled equimolarly,

575 according to species and geographic location; e.g., the purified PCR products of five

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576 Fucus vesiculosus individuals from Norway were pooled into one sample before

577 sequencing library preparation.

578 Sequencing libraries were prepared using the TruSeq Nano DNA Library

579 Preparation Kit at The Natural History Museum, London UK, following the

580 manufacturer’s protocol. High throughput sequencing was conducted on a MiSeq v3

581 flow cell using 2x300 bp paired-end reads. Base calling was performed by Illumina

582 Real Time Analysis v1.18.54 and was demultiplexed and converted to FastQ files with

583 Illumina Bcl2fastq v1.8.4.

584 The complete sequencing dataset is available at the European Nucleotide

585 Archive under the study accession number XXXXXXXX (http://www.ebi.ac.uk/ena/

586 data/view/ XXXXXXXX).

587 To obtain full length 18S sequences of the brown algal host species, three DNA

588 isolates from each brown algal species were subjected to PCR amplification using the

589 general eukaryotic 18S primers NSF83 (5’-GAAACTGCGAATGGCTCATT-3’ [120])

590 and 1528R (5’-TCCTTCTGCAGGTTCACCTAC-3’ [121]) utilising illustra™

591 PuReTaq Ready-To-Go™ PCR beads (GE Healthcare). PCR reactions contained 1

592 Illustra PuReTaq Ready-To-Go-bead, 1μl of DNA template, 0.5 μM of each primer,

593 filled to 25μl with water. PCR settings were: initial denaturation at 94°C for 2 min; 35

594 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 30 s, elongation at 68°C

595 for 1.5 min; final elongation step at 68°C for 15 min. The PCR products were cloned

596 using the TOPO TA Cloning Kit for Sequencing (Invitrogen), according to the

597 manufacturer’s protocol. The clones were grown overnight in Luberia-Bertoni (LB)

598 media amended with 50 μg/mL ampicillin. From each brown algal species, 20-30

599 bacterial colonies/cloned fragments, were subjected to PCR reactions with the vector

600 primers T7 and M13R and using ca 0.5 μl of the bacterial suspension as template. PCR

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601 settings were: initial denaturation at 94°C for 10 min; 30 cycles of denaturation at 94°C

602 for 30 s, annealing at 53°C for 1 min, elongation at 72°C for 2 min; final elongation

603 step at 72°C for 10 min. The PCR products were cleaned using illustra™ ExoProStar

604 1-Step (GE Healthcare) and then Sanger sequenced (GATC Eurofins Genomics,

605 Germany).

606

607 Bioinformatics

608 The sequence data was processed using a workflow for MiSeq amplicon data

609 (https://github.com/ramalok/amplicon_processing). In short, prior to downstream

610 analyses error correction was done with BayesHammer [122], before merging the

611 paired-end reads with PEAR [123]. Thereafter, quality filtering, length check and fasta

612 conversion (from fastq to fasta) was done with USEARCH [124] before reads were put

613 into the correct 5’ – 3’ direction using profile hidden Markov models (hmmer-3.0rc2).

614 Dereplication, sorting by abundance and discarding of singletons was done using

615 USEARCH 64bit version. Clustering of reads into Operational Taxonomic Units

616 (OTUs) was done using the UPARSE algorithm as implemented in USEARCH,

617 applying a 97% sequence similarity threshold. De-novo and reference-based chimera

618 checking was performed and chimeric sequences discarded. OTUs were taxonomically

619 assigned using BLASTn against the PR2 v. 4.12.0 [125], GenBank [126] and SILVA

620 SSU [127] databases. We used a relaxed BLAST search strategy (BLASTn parameters:

621 -evalue 0.0001 -perc_identity 75) to retrieve distant sequences, and only retrieved the

622 best hit per query sequence. The PR2 taxonomy was used for taxonomy assignment for

623 the majority of the following analyses.

624

625 “Post-UPARSE trimming” & Diversity analyses

25 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

626 Species (OTU) composition analyses and further trimming of the dataset was done

627 using R v. 3.6.3 (R Core Team 2013). As we expected the sequence dataset was heavily

628 dominated by brown algal host reads, and we did therefore not normalise the dataset

629 because this would have led to removal of true diversity since most of the other OTUs

630 had very low read numbers compared to the brown algal OTUs. Further, all OTUs

631 assigned to Metazoa were removed from the dataset in addition to OTUs assigned to

632 land plants. The 57 OTUs (16.3% of the total number of OTUs) that matched those of

633 Metazoa are shown in Supplementary Figure S5, Supplementary Table S5.The trimmed

634 dataset, containing only non-metazoan eukaryotes, was used for further analyses.

635 The taxonomic composition of OTUs was visualized using the ggplot2 package

636 v. 3.3.2. [128]. To investigate how the composition of (micro)eukaryotes associated

637 with the different brown algal hosts, Bray-Curtis pairwise distances were calculated

638 using vegan v. 2.5-6 [129] for all brown algal samples (including/excluding brown algal

639 OTUs) and visualised using non-metric multidimensional scaling (NMDS) cluster

640 analyses (ggplot2 package v. 3.3.2 [128]). Analysis of similarities [ANOSIM; 130] was

641 calculated using vegan v. 2.5-6 [129] to test if there was a statistical significant

642 differences between clusters of libraries corresponding to different brown algal host

643 genera. The similarity percentage [SIMPER; 130] was calculated using the

644 simper.pretty.R script [131] to identify OTUs that contributed the most to the

645 dissimilarity observed. Indicator species analyses (IndVal) were carried out using the

646 indicspecies package v. 1.7.9. [132], to identify OTUs most preferentially associated

647 with specific brown algal host genera. Venn diagrams intersects were computed using

648 the function overLapper and plotted using the function vennPlot in the R-package

649 systemPipeR v. 1.24.3 [133].

650

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651 Phylogenetic analysis

652 All OTUs were places into a global eukaryotic alignment of near full-length 18S

653 reference sequence using MAFFT v7.300b with the ‘einsi’ option for global homology

654 and long gaps for the reference sequences [134]. The shorter OTUs from the V4 region

655 (300-450 bp) were added to the reference alignment using the –addfragment option in

656 MAFFT and ambiguously aligned sites were removed with TrimAl [135] with –gt 0.3

657 and –st 0.001. Phylogenetic trees were built with RAxML v8.0.26 under the

658 GTRGAMMA model and the autoMRE bootsopping criteria [136]. Based on a

659 combination of the phylogenetic affinities of the OTUs and the taxonomic assignment

660 from PR2, individual phylogenies of the most abundant/diverse groups of

661 microeukaryotes – and those with known associations with brown algae - were

662 constructed: i.e Fungi, Oomycetes & labyrinthulids, Cercozoa, diatoms, red, brown,

663 and green algae, ciliates, other alveolates, Amoebozoa, and centrohelid heliozoans.

664 Individual sequence datasets were constructed including OTUs from each group

665 whose taxonomic annotation and phylogenetic position were concordant, and their

666 closest Blastn matches from GenBank. Sequence alignments were carried out in

667 MAFFT as described above. Phylogenetic trees were built with MrBayes v.3.2.6 [137].

668 Two separate MC3 runs with randomly generated starting trees were carried out for 4

669 million generations each with 1 cold and 3 heated chains. The evolutionary model

670 applied a GTR substitution matrix, with a 4-category autocorrelated gamma correction.

671 All parameters were estimated from the data. The trees were sampled every 1000

672 generations and the first 1 million generations discarded as burn-in. All phylogenetic

673 analyses were carried out on the Cipres server [138]. Heatmaps were made for all

674 phylogenetic trees to display the proportional read abundance (log10) for each OTU in

675 the different samples.

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676

677 Declarations

678 Ethics approval and consent to participate

679 Not applicable

680 Consent for publication

681 Not applicable

682

683 Availability of data and materials

684 The dataset generated are available in XX repository, under the accession XXXX-

685 XXXX (link to dataset).

686

687 Competing interest

688 The authors declare that they have no competing interests

689

690 Funding

691 This study has been supported financially by research funds “Sunniva og Egil

692 Baardseths legat, til støtte for forskning på makroalger” and from the University of Oslo

693 and the Natural History Museum, London to MFMB.

694

695 Authors’ contributions

696

697 Acknowledgements

698 We thank Jonas Thormar and Jens Ådne Haga for help with field sampling of brown

699 algae in Oslofjord. Drøbak Biological Station and Hans Erik Karlsen are thanked for

700 providing sampling equipment and laboratory facilities for sample preparations.

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

701

702

703 Figure titles and figure legends

704 Figure 1. Diversity of eukaryotes in brown algal holobionts.

705 Proportional abundance of OTUs taxonomically assigned to eukaryotic major

706 Kingdoms or ‘supergroup’ in the brown algal samples of Fucus serratus, F.

707 vesiculosus, Ascophyllum nodosum, Himanthalia elongata, Saccorhiza polyschides,

708 Laminaria digitata and Saccharina latissima. The sampling location for each brown

709 alga is shown in parenthesis; NO = Norway and UK = The United Kingdom, together

710 with the total number of OTUs per library. The asterisk (NO*) represent F. vesiculosus

711 sampled in Norway, May 2013. All other samples were collected in October 2015.

712

713 Figure 2. Abundance of eukaryotic taxonomic groups.

714 Percentage representation of OTUs assigned to the different taxonomic groups at (A)

715 Kingdom or ‘supergroup’ level and at lower taxonomic level (B) in all of the brown

716 algal samples combined. To see the percentage of reads and percentage of OTUs for all

717 the taxonomic groups per brown algal sample, see Supplementary table S2.

718

719 Figure 3. Oomycetes and Labyrinthulids phylogeny with heatmap representing

720 proportional read abundance (log10) of OTUs per sample. Bayesian phylogeny of

721 Oomycetes and Labyrinthulomycetes with bootstrap values from maximum likelihood

722 analysis added. Black circles represent maximal support (posterior probability = 1, and

723 bootstrap support = 100%). OTUs from this study are shown in bold. The percentage

724 identity to the most similar reference sequence is shown in parenthesis after each OTU.

725 Host or type of environment the reference sequences were retrieved from in previous

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

726 studies are listed in parenthesis after each GenBank accession number. The scale bar

727 represents 0.2 substitutions per site. Abbreviations used for descriptions of

728 environment: env.S = environmental sample, marine sediment; env.W = environmental

729 sample, marine water; env.FW = environmental sample, fresh water; env.So =

730 environmental sample, soil. The heatmap illustrates the log10 read abundance for each

731 OTU in the brown algal samples of Fucus serratus, F. vesiculosus, Ascophyllum

732 nodosum, Himanthalia elongata, Saccorhiza polyschides, Laminaria digitata and

733 Saccharina latissima. The sampling location for each brown alga is shown in

734 parenthesis; NO = Norway and UK = The United Kingdom. All samples were collected

735 in October 2015, except F. vesiculosus (NO*) which was sampled in Norway, May

736 2013.

737

738 Figure 4. Cercozoa: phylogeny with heatmap representing proportional read

739 abundance (log10) of OTUs per sample. Bayesian phylogeny of Cercozoa with

740 bootstrap values from maximum likelihood analysis added. Black circles represent

741 maximal support (posterior probability = 1, and bootstrap support = 100%). OTUs from

742 this study are shown in bold. The percentage identity to the most similar reference

743 sequence is shown in parenthesis after each OTU. Host or type of environment the

744 reference sequences were retrieved from in previous studies are listed in parenthesis

745 after each GenBank accession number. The scale bar represents 0.1 substitutions per

746 site. Abbreviations used for descriptions of environment: env.S = environmental

747 sample, marine sediment; env.W = environmental sample, marine water; env.FW =

748 environmental sample, fresh water; env.BW = environmental sample, brackish water;

749 env.S/W = environmental sample, marine sediment or water; env.So = environmental

750 sample, soil. The heatmap illustrates the log10 read abundance for each OTU in the

30 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

751 brown algal samples of Fucus serratus, F. vesiculosus, Ascophyllum nodosum,

752 Himanthalia elongata, Saccorhiza polyschides, Laminaria digitata and Saccharina

753 latissima. The sampling location for each brown alga is shown in parenthesis; NO =

754 Norway and UK = The United Kingdom. All samples were collected in October 2015,

755 except F. vesiculosus (NO*) which was sampled in Norway, May 2013.

756

757 Figure 5. Fungi: phylogeny with heatmap representing proportional read

758 abundance (log10) of OTUs per sample. Bayesian phylogeny of Fungi with bootstrap

759 values from maximum likelihood analysis added. Black circles represent maximal

760 support (posterior probability = 1, and bootstrap support = 100%). OTUs from this

761 study are shown in bold. The percentage identity to the most similar reference sequence

762 is shown in parenthesis after each OTU. Host or type of environment the reference

763 sequences were retrieved from in previous studies are listed in parenthesis after each

764 GenBank accession number. The scale bar represents 0.07 substitutions per site.

765 Abbreviations used for descriptions of environment: env.S = environmental sample,

766 marine sediment; env.W = environmental sample, marine water; env.FW =

767 environmental sample, fresh water; env.So = environmental sample, soil; env.A =

768 environmental sample, air. The heatmap illustrates the log10 read abundance for each

769 OTU in the brown algal samples of Fucus serratus, F. vesiculosus, Ascophyllum

770 nodosum, Himanthalia elongata, Saccorhiza polyschides, Laminaria digitata and

771 Saccharina latissima. The sampling location for each brown alga is shown in

772 parenthesis; NO = Norway and UK = The United Kingdom. All samples were collected

773 in October 2015, except F. vesiculosus (NO*) which was sampled in Norway, May

774 2013.

775

31 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

776 Figure 6. Bacillariophyceae: phylogeny with heatmap representing proportional

777 read abundance (log10) of OTUs per sample. Bayesian phylogeny of diatoms with

778 bootstrap values from maximum likelihood analysis added. Black circles represent

779 maximal support (posterior probability = 1, and bootstrap support = 100%). OTUs from

780 this study are shown in bold. The percentage identity to the most similar reference

781 sequence is shown in parenthesis after each OTU. Host or type of environment the

782 reference sequences were retrieved from in previous studies are listed in parenthesis

783 after each GenBank accession number. The scale bar represents 0.06 substitutions per

784 site. Abbreviations used for descriptions of environment: env.S = environmental

785 sample, marine sediment; env.W = environmental sample, marine water; env.Si =

786 environmental sample, sea ice; GC = gut content. The heatmap illustrates the log10 read

787 abundance for each OTU in the brown algal samples of Fucus serratus, F. vesiculosus,

788 Ascophyllum nodosum, Himanthalia elongata, Saccorhiza polyschides, Laminaria

789 digitata and Saccharina latissima. The sampling location for each brown alga is shown

790 in parenthesis; NO = Norway and UK = The United Kingdom. All samples were

791 collected in October 2015, except F. vesiculosus (NO*) which was sampled in Norway,

792 May 2013.

793

794 Figure 7. Brown-Red-Green: phylogeny with heatmap representing proportional

795 read abundance (log10) of OTUs per sample. Bayesian phylogeny of Phaeophyceae,

796 Rhodophyta and Chlorophyta with bootstrap values from maximum likelihood analysis

797 added. OTUs from this study are shown in bold. The percentage identity to the most

798 similar reference sequence is shown in parenthesis after each OTU. Host or type of

799 environment the reference sequences were retrieved from in previous studies are listed

800 in parenthesis after each GenBank accession number. The longer 18S sequences

32 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

801 generated by cloning and sanger sequencing in this study are shown in bold and

802 includes which brown algal host species they were amplified from. The brown algal

803 endophytes isolated and sequenced by Attwood (2005) are all in the ‘brown

804 endophytes’ clade and are shown in capital letters. The scale bar represents 0.7

805 substitutions per site. The heatmap illustrates the log10 read abundance for each OTU

806 in the brown algal samples of Fucus serratus, F. vesiculosus, Ascophyllum nodosum,

807 Himanthalia elongata, Saccorhiza polyschides, Laminaria digitata and Saccharina

808 latissima. The sampling location for each brown alga is shown in parenthesis; NO =

809 Norway and UK = The United Kingdom. All samples were collected in October 2015,

810 except F. vesiculosus (NO*) which was sampled in Norway, May 2013.

811

812 Figure 8. Brown algal endophytes

813 Light microscopy images of phaeophyte endophytes isolated from Fucus serratus,

814 Mastocarpus stellatus, Chorda filum, Osmundea pinnatifida, and Chondrus crispus. (I)

815 Streblonema sp.: A – terminal sporangium, showing apical spore release; B – branching

816 thallus from spherical cells; C – growth pattern of new filament; D – discoid parietal

817 plastid. (II) Myrionema strangulans: A – Phaeophyceae hair; B – plurilocular

818 sporangia; C- two different cell shapes; D – unilocular lateral sporangium. (III)

819 Microspongium tenuisimum: A - plurilocular sporangia releasing zoospores; B -

820 Phaeophyceae hair; C – bifurcating branch; D – ovate cell, showing plate-like plastid

821 with prominent pyrenoids; E – waisted cross cell wall. (IV) Mytriotrichia clavaeformis:

822 A - Phaeophyceae hair; B – clubbed terminal cell; C - unilocular clustering sporangia;

823 D – empty clubbed terminal sporangium. (V) Ectocarpus sp.: A – ribbon-shaped

824 chloroplast; B – pyrenoid; C – pseudohairs; D – pseudohairs shoing growth from

33 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

825 filament; E – branching growth form. (VI) Chorda filum: A – densely pigmented cells;

826 B – prostrate clumps of branched filaments.

827

828 Figure 9. Community composition. Non metric multidimensional scaling (NMDS)

829 analysis of the composition of eukaryotic OTUs in the brown algal samples based on

830 Bray-Curtis pairwise distances. (A) including brown algal/Phaeophyceae OTUs and

831 (B) excluding brown algal/Phaeophyceae OTUs.

832

833 Figure 10. Shared and unique OTUs in the brown algal orders.

834 Venn diagrams displaying the number of shared and unique OTUs between the brown

835 algal host orders Fucales, Laminariales and Tilopteridales. To see the taxonomic

836 assignment for the shared OTUs see Table 4, and for the unique OTUs see

837 Supplementary Table S4.

838

839

840 Supplementary figures:

841

842 Supplementary figure 1. Ciliates: phylogeny with heatmap representing

843 proportional read abundance (log10) of OTUs per sample. Bayesian phylogeny of

844 Ciliates with bootstrap values from maximum likelihood analysis added. Black circles

845 represent maximal support (posterior probability = 1, and bootstrap support = 100%).

846 OTUs from this study are shown in bold. The percentage identity to the most similar

847 reference sequence is shown in parenthesis after each OTU. Host or type of

848 environment the reference sequences were retrieved from in previous studies are listed

849 in parenthesis after each GenBank accession number. The scale bar represents 0.2

34 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

850 substitutions per site. Abbreviations used for descriptions of environment: env.S =

851 environmental sample, marine sediment; env.W = environmental sample, marine water;

852 env.FW = environmental sample, fresh water; env.WW = environmental sample, waste

853 water; env.BW = environmental sample, brackish water; env.S/W = environmental

854 sample, marine sediment or water; env.MBF = environmental sample, marine biofilm;

855 env.FBF = environmental sample, freshwater biofilm; env.So = environmental sample,

856 soil. The heatmap illustrates the log10 read abundance for each OTU in the brown algal

857 samples of Fucus serratus, F. vesiculosus, Ascophyllum nodosum, Himanthalia

858 elongata, Saccorhiza polyschides, Laminaria digitata and Saccharina latissima. The

859 sampling location for each brown alga is shown in parenthesis; NO = Norway and UK

860 = The United Kingdom. All samples were collected in October 2015, except F.

861 vesiculosus (NO*) which was sampled in Norway, May 2013.

862

863 Supplementary figure 2. Apicomplexa: phylogeny with heatmap representing

864 proportional read abundance (log10) of OTUs per sample. Bayesian phylogeny of

865 Apicomplexa and perkinsids with bootstrap values from maximum likelihood analysis

866 added. Black circles represent maximal support (posterior probability = 1, and bootstrap

867 support = 100%). OTUs from this study are shown in bold. The percentage identity to

868 the most similar reference sequence is shown in parenthesis after each OTU. Host or

869 type of environment the reference sequences were retrieved from in previous studies

870 are listed in parenthesis after each GenBank accession number. The scale bar represents

871 0.3 substitutions per site. Abbreviations used for descriptions of environment: env.S =

872 environmental sample, marine sediment; env.W = environmental sample, marine water;

873 env.FW = environmental sample, fresh water; env.I = environmental sample, ice; GC

874 = gut content. The heatmap illustrates the log10 read abundance for each OTU in the

35 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

875 brown algal samples of Fucus serratus, F. vesiculosus, Ascophyllum nodosum,

876 Himanthalia elongata, Saccorhiza polyschides, Laminaria digitata and Saccharina

877 latissima. The sampling location for each brown alga is shown in parenthesis; NO =

878 Norway and UK = The United Kingdom. All samples were collected in October 2015,

879 except F. vesiculosus (NO*) which was sampled in Norway, May 2013.

880

881 Supplementary figure 3. Amoebozoa and Centroheliozoa phylogeny with heatmap

882 representing proportional read abundance (log10) of OTUs per sample. Bayesian

883 phylogeny of Amoebozoa and Centroheliozoa with bootstrap values from maximum

884 likelihood analysis added. Black circles represent maximal support (posterior

885 probability = 1, and bootstrap support = 100%). OTUs from this study are shown in

886 bold. The percentage identity to the most similar reference sequence is shown in

887 parenthesis after each OTU. Host or type of environment the reference sequences were

888 retrieved from in previous studies are listed in parenthesis after each GenBank

889 accession number. The scale bar represents 0.2 and 0.08 substitutions per site for

890 Amoebozoa and Centroheliozoa respectively. Abbreviations used for descriptions of

891 environment: env.FWS = environmental sample, freshwater sediment; env.W =

892 environmental sample, marine water; env.So = environmental sample, soil. The

893 heatmap illustrates the log10 read abundance for each OTU in the brown algal samples

894 of Fucus serratus, F. vesiculosus, Ascophyllum nodosum, Himanthalia elongata,

895 Saccorhiza polyschides, Laminaria digitata and Saccharina latissima. The sampling

896 location for each brown alga is shown in parenthesis; NO = Norway and UK = The

897 United Kingdom. All samples were collected in October 2015, except F. vesiculosus

898 (NO*) which was sampled in Norway, May 2013.

899

36 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

900 Supplementary figure 4. Dinoflagellates: phylogeny with heatmap representing

901 proportional read abundance (log10) of OTUs per sample. Bayesian phylogeny of

902 Dinoflagellates with bootstrap values from maximum likelihood analysis added. Black

903 circles represent maximal support (posterior probability = 1, and bootstrap support =

904 100%). OTUs from this study are shown in bold. The percentage identity to the most

905 similar reference sequence is shown in parenthesis after each OTU. Host or type of

906 environment the reference sequences were retrieved from in previous studies are listed

907 in parenthesis after each GenBank accession number. The scale bar represents 0.08

908 substitutions per site. Abbreviations used for descriptions of environment: env.S =

909 environmental sample, marine sediment; env.W = environmental sample, marine water;

910 env.BW = environmental sample, brackish water. The heatmap illustrates the log10 read

911 abundance for each OTU in the brown algal samples of Fucus serratus, F. vesiculosus,

912 Ascophyllum nodosum, Himanthalia elongata, Saccorhiza polyschides, Laminaria

913 digitata and Saccharina latissima. The sampling location for each brown alga is shown

914 in parenthesis; NO = Norway and UK = The United Kingdom. All samples were

915 collected in October 2015, except F. vesiculosus (NO*) which was sampled in Norway,

916 May 2013.

917

918 Supplementary figure 5. Diversity of metazoan OTUs detected in brown algal

919 holobionts.

920 Proportional abundance of OTUs taxonomically assigned to Metazoa in the brown algal

921 samples of Fucus serratus, F. vesiculosus, Ascophyllum nodosum, Himanthalia

922 elongata, Saccorhiza polyschides, Laminaria digitata and Saccharina latissima. The

923 sampling location for each brown alga is shown in parenthesis; NO = Norway and UK

37 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

924 = The United Kingdom. The asterisk (NO*) represent F. vesiculosus sampled in

925 Norway, May 2013. All other samples were collected in October 2015.

926

927

928 References:

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Table 1. Summary of pairwise SIMPER analyses between brown algal host samples grouped at genus level. Showing the OTUs that contributed to more than 10% of the observed dissimilarity between host genera according to SIMPER, when Phaeophyceae OTUs were included in the analyses. OTUs contributing >10 % to the dissimilarity between brown algal hosts

(Including Phaeophyceae OTUs) Phylum Stramenopiles Class Phaeophyceae PR2 Silvetia Fucus Fucus Silvetia Saccorhiza Agarum clathratum taxonomy siliquosa gardneri gardneri siliquosa polyschides

Inferred host Ascophyllum Fucus Fucus Himanthalia Saccorhiza Laminariales

taxonomy nodosum spp. vesiculosus elongata polyschides (L. digitata + S. latissima) SIMPER comparison Dissimilarity OTU 2 OTU 49 OTU 41 OTU 5 OTU 3 OTU 1 (%) Fucus – Ascophyllum 74.4 41.3 8.4 4.8 Fucus – Himanthalia 99.8 20.0 5.1 9.6 36.6 Fucus – Saccorhiza 97.9 21.1 5.4 10.1 35.8 Fucus – Laminaria 96.7 21.0 5.4 10.1 38.3 Fucus – Saccharina 97.3 15.2 3.9 7.4 54.3 Ascophyllum – Himanthalia 99.9 54.9 11.7 19.7 Ascophyllum – Saccorhiza 99.8 56.6 12.1 18.7 Ascophyllum – Laminaria 99.9 56.4 12.1 20.8 Ascophyllum – Saccharina 99.9 47.3 10.1 33.3 Himanthalia – Saccorhiza 99.7 45.0 41.6 Himanthalia – Laminaria 99.7 44.7 45.8 Himanthalia – Saccharina 99.8 31.3 61.6 Saccorhiza – Laminaria 98.3 44.2 49.1 Saccorhiza – Saccharina 98.5 30.4 64.6 Laminaria – Saccharina 31.4

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Table 2. Summary of pairwise SIMPER analyses between brown algal host samples grouped at genus level. Showing the OTUs that contributed to more than 10% of the observed dissimilarity between host genera according to SIMPER, when Phaeophyceae OTUs were excluded from the analyses. OTUs contributing >10 % to the dissimilarity between brown algal hosts

(Excluding Phaeophyceae OTUs) Phylum Chlorophyta Rhodophyta Fungi Stramenopiles Unassigned Class Ulvophyceae Florideo- Basidiomycota Ascomycota Oomycota NA phycaea

PR2 tax Ulvophyceae Ceramium Agarico- Exobasidio- Torulaspora Pythium NA sp. rubrum mycetes sp. mycetes sp. franciscae chamaihyphon

SIMPER comparison Dissi- OTU 6 OTU 21 OTU 57 OTU 25 OTU 50 OTU 42 OTU 61 milarity (%) Fucus – Ascophyllum 95.9 33.2 15.7 11.9 4.2 Fucus – Himanthalia 98.5 44.9 4.8 Fucus – Saccorhiza 94.0 32.6 5.1 5.8 Fucus – Laminaria 98.5 43.0 3.2 Fucus – Saccharina 98.7 41.3 12.2 Ascophyllum – Himanthalia 99.4 41.3 31.1 10.5 4.4 Ascophyllum – Saccorhiza 83.3 13.8 15.9 5.4 7.7 Ascophyllum – Laminaria 92.9 35.9 28.6 9.8 Ascophyllum – Saccharina 98.6 36.4 12.8 27.0 9.2 Himanthalia – Saccorhiza 98.7 13.7 14.2 4.2 Himanthalia – Laminaria 96.5 11.1 3.0 28.6 Himanthalia – Saccharina 98.1 58.2 1.2 17.5 Saccorhiza – Laminaria 91.9 10.4 13.3 Saccorhiza – Saccharina 98.6 12.2 12.2 12.5 Laminaria – Saccharina 97.7 7.4 47.2

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Table 3. Indicator species analysis (IndVal) showing OTUs most preferentially associated with the brown algal hosts. OTU# Associated with p-value Taxonomic assignment OTU 149 Ascophyllum 0.0185 * Stramenopiles; Ochrophyta; Phaeophyceae; Silvetia; Silvetia siliquosa OTU 381 Ascophyllum 0.0175 * Stramenopiles; Ochrophyta; Phaeophyceae; Silvetia; Silvetia siliquosa OTU 244 Ascophyllum 0.0005 *** Stramenopiles; Ochrophyta; Phaeophyceae; Silvetia; Silvetia siliquosa OTU 2 Ascophyllum 0.0020 ** Stramenopiles; Ochrophyta; Phaeophyceae; Silvetia; Silvetia siliquosa OTU 49 Ascophyllum 0.0127 * Stramenopiles; Ochrophyta; Phaeophyceae; Fucus; Fucus gardneri OTU 25 Ascophyllum 0.0446 * Fungi; Basidiomycota; Ustilaginomycotina; Exobasidiomycetes; Exobasidiomycetes_X sp. OTU 1 Laminaria + Saccharina 0.0033 ** Stramenopiles; Ochrophyta; Phaeophyceae; Agarum; Agarum clathratum The Associated with column show which library/host species the OTUs were preferentially associated with. Abbreviations used; Ascophyllum = Ascophyllum nodosum, Laminaria = Laminaria digitata, Saccharina = Saccharina latissima. The asterisk associated with IndVal p-values indicat significance at ≤ 0.001 (***), ≤ 0.01 (**), ≤ 0.05 (*).

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Table 4. Taxonomy of the shared OTUs between the three brown algal orders: Fucales, Laminariales and Tilopteridales, as shown in Venn plot (Fig. 10). Overlap OTU# Taxonomic assignment

Fucales + OTU 256 Alveolata Ciliophora Spirotrichea Laminariales + OTU 10, OTU 6 Archaeplastida Chlorophyta Ulvophyceae Tilopteridales OTU 21 Archaeplastida Rhodophyta Florideophyceae (25 OTUs) OTU 25 Opisthokonta Fungi Basidiomycota OTU 94, OTU 233, OTU 234, OTU 68, Stramenopiles Ochrophyta Bacillariophyta OTU 112 OTU 1, OTU 236, OTU 28, OTU 292, Stramenopiles Ochrophyta Phaeophyceae OTU 41, OTU 49, OTU 146, OTU 70, OTU 4, OTU 7, OTU 9, OTU 3, OTU 2, OTU 348, OTU 5 Fucales + OTU 111, OTU 148, OTU 137, OTU Alveolata Ciliophora Oligohymenophorea, Phyllopharyngea, Spirotrichea Tilopteridales 188, OTU 151 (22 OTUs) OTU 298 Amoebozoa Lobosa Vermistella sp. OTU 180, OTU 87 Archaeplastida Chlorophyta Ulvophyceae OTU 110 Rhizaria Cercozoa Endomyxa OTU 265, OTU 197, OTU 220, OTU 72, Stramenopiles Ochrophyta Bacillariophyta OTU 63, OTU 237, OTU 17 OTU 300 Stramenopiles Ochrophyta Chrysophyceae OTU 105 Stramenopiles Ochrophyta Phaeophyceae OTU 38, OTU 42 Stramenopiles Pseudofungi Oomycota OTU 166, OTU 173 Unassigned NA NA Fucales + OTU 89, OTU 97 Alveolata Ciliophora Oligohymenophorea, Phyllopharyngea Laminariales OTU 107, OTU 191 Alveolata Dinoflagellata Dinophyceae (20 OTUs) OTU 69 Archaeplastida Chlorophyta Ulvophyceae OTU 226, OTU 118, OTU 257 Opisthokonta Fungi Ascomycota, Basidiomycota OTU 93, OTU 172 Stramenopiles Ochrophyta Bacillariophyta OTU 23, OTU 26, OTU 389, OTU 149, Stramenopiles Ochrophyta Phaeophyceae OTU 244, OTU 326, OTU 381 OTU 238 Stramenopiles Opalozoa MAST-3 OTU 295 Stramenopiles Pseudofungi Oomycota OTU 249 Stramenopiles Sagenista Labyrinthulomycetes Laminariales + OTU 193, OTU 74 Alveolata Dinoflagellata Dinophyceae Tilopteridales OTU 168 Stramenopiles Sagenista Labyrinthulomycetes (3 OTUs) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table 5. Hosts with brown algal endophytes selected for isolation from the B. Rinkel collection at the Natural History Museum, London.

Sample ID Host Site name, county Date Collector BRE1sn1 Chondrus crispus Sheerness, Kent 22.10.03 J. Brodie BRE9ls2 Chondrus crispus Lilstock, Somerset 27.10.03 B. Rinkel BRE38sm11 Mastocarpus stellatus Sidmouth, Devon 28.10.01 B. Rinkel BRE74ls1 Fucus serratus Lilstock, Somerset 27.10.03 B. Rinkel BRE98sm12 Mastocarpus stellatus Sidmouth, Devon 28.10.01 B. Rinkel BRE117sm22 Osmundea osmunda Sidmouth, Devon 28.10.01 B. Rinkel BRE118sm22 Osmundea osmunda Sidmouth, Devon 28.10.01 B. Rinkel BRE128hi1 Osmundea pinnatifida Hayling Island, Hampshire 05.02.04 B. Rinkel BRE152cb2 Chondrus crispus Castle Beach, Pembrokeshire 09.03.04 B. Rinkel BRE157br1 Mastocarpus stellatus Black Rock, Pembrokeshire 10.03.04 B. Rinkel E026pz Chondrus crispus Polzeath, Cornwall 19.06.04 M. Holmes E030Nb Mastocarpus stellatus Norway 23.06.04 J. Brodie E326wr Fucus serratus West Runton, Norfolk 28.09.04 B. Rinkel E360sm2 Chorda filum Sidmouth, Devon 28.10.03 B. Rinkel E361ac Chorda filum A’Chleit, Bute 10.07.04 B. Rinkel E365sg Mastocarpus stellatus Seagreens, Aberdeenshire 16.07.04 B. Rinkel E368 Chondrus crispus Bouley Bay, Jersey 13.03.05 B. Rinkel E370 Chondrus crispus Bouley Bay, Jersey 13.03.05 B. Rinkel E377 Chondrus crispus Greve de Lacq, Jersey 14.03.05 B. Rinkel LTE19/1 Osmundea pinnatifida Black Rock, Pembrokeshire 10.03.04 B. Rinkel

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100%

Microeukaryotes

Alveolata 75% Amoebozoa Apusozoa Chlorophyta Choanoflagellida

50% Fungi Hacrobia Mesomycetozoa Rhizaria

Proportional OTU abundance Rhodophyta 25% Stramenopiles Unassigned

0% L . digitata (UK, 27) L . digitata (NO, 55) F . serratus (UK, 60) S . latissima (NO, 36) A . nodosum (NO, 41) H . elonogata (UK, 26) F . vesiculosus (UK, 85) F . vesiculosus (NO, 98) S . polyschides (UK, 90) F . vesiculosus (NO*, 101) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Stramenopiles 42.6 %

Alveolata 28.3 %

Cercozoa (Rhizaria) 6.0 %

Fungi 7.9 %

Archaeplastida 5.3 %

Hacrobia 3.4 %

Amoebozoa 2.3 %

Apusozoa 1.1 %

Choanoflagellida 1.1 %

Mesomycetozoa 0.4 %

Unassigned 1.5 %

0 10 20 30 40 % OTUs

Phaeophyceae 18.9 % Diatomea 11.7 % Labyrinthulomycetes 6.8 % Oomycota 2.6 % Dictyochophyceae 1.1 % Chrysophyceae 0.8 % MAST 0.8 % Ciliophora 18.5 % Dinoflagellata 6.0 % Apicomplexa 3.4 % Perkinsea 0.4 % Endomyxa 2.6 % Filosa−Thecofilosea 1.5 % Endomyxa−Phytomyxea 0.8 % Filosa−Imbricatea 0.8 % Filosa−Sarcomonadea 0.4 % Basidiomycota 3.8 % Chytridiomycota 2.3 % Ascomycota 1.9 % Chlorophyta 3.4 % Rhodophyta 1.9 % Centroheliozoa 3.0 % Katablepharidophyta 0.4 % Lobosa 1.5 % Evosea 0.4 % Breviatea 0.4 % Apusomonadidae 1.1 % Craspedida 1.1 % Ichthyosporea 0.4 % Unassigned 1.5 % 0 5 10 15 20 % OTUs bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A . nodosum (NO) H . elonogata (UK) serratus (UK) F . serratus (NO*) F . vesiculosus (UK) F . vesiculosus L . digitata (NO) L . digitata (UK) S . latissima (NO) vesiculosus (NO) F . vesiculosus S . polyschides (UK)

0.99 FJ389817 Fav2 R2 183F (coral) OTU 307 (99.8%) OTU 307 0.9 FJ389878 Fav18 4 490F (coral) OTU 308 (93.8%) OTU 308 EF100267 D2P04C03 (env.S) OTU 153 0.97 OTU 153 (99.3%) LM653283 Aplanochytrium sp. ANT10 (brown algae) 0.99 FJ153754 (seagrass) 0.79 OTU 272 (97.2%) OTU 272 OTU 168 (95.7%) OTU 168 KX160007 Aplanochytrium blankum (sea star) OTU 160 (97.6%) OTU 160 EF100254 D2P04F11 (env.S) OTU 161 (99.8%) OTU 161 OTU 208 (85.3%) Labyrinthulida OTU 208 EU431329 Labyrinthula sp. LTH (amoeba) OTU 221 (89.3) OTU 221 AB973544 Aplanochytrium sp. SEK 717 (env.W) 0.95 KP685309 F 13 1105 (env.S) OTU 251 (99.8%) OTU 251

Labyrinthulomycetes 0.8 FJ800615 PBW102907 (env.W) OTU 249 (98.3%) OTU 249 AF265339 Labyrinthuloides minuta (culture) OTU 402 (86.6%) OTU 402 OTU 71 (89.6%) OTU 71 KT812806 A8 28665 (env.S) AB973557 Thraustochytriidae SEK 702 (env.W) AB973547 Thraustochytriidae SEK 692 (env.W) OTU 339 (87.4%) OTU 339 KX965681 Quahog parasite QPX (clam) OTU 116 (84.1%) OTU 116 EF100252 D2P04F01e (env.S)

OTU 195 (99.8%) Thraustochytrida OTU 195 GQ354272 Oblongichytrium sp. PBS03 (env.S) 0.92 EF100297 D3P06D06 (env.S) 0.86 OTU 38 (99.5%) OTU 38 KU764783 Anisolpidium rosenvingei (brown algae) 0.9 KU764786 Anisolpidium ectocarpii (brown algae) 0.9 OTU 295 (99.8%) OTU 295 0.84 OTU 214 (90.9%) OTU 214 0.97 KM210530 Olpidiopsis feldmanni (red algae) 0.82 OTU 126 (99%) OTU 126

KT811839 53c 48093 (env.S) Olpidiopsidales

Oomycetes OTU 42 (99.8%) OTU 42 EF023544 Amb 18S 1146 (env.So) 0.97 KT273921 Lagenisma coscinodisci (diatom) OTU 290 (92.4%) OTU 290 AY598666 Phytopythium chamaehyphon (culture) 0.78 KP685311 F13 1124 (env.S) OTU 312 (98.4%) 0.82 OTU 312

AB178865 Halioticida noduliformans NJM0034 (abalone) Perenosporales & Halioticida 0.92 AY046668 CS E036 (env.S) OTU 338 (98.8%) OTU 338 AF265331 Diplophrys marina AY821979 CV1 B1 3 (env.FW) OTU 142 (92.9%) OTU 142 AB856528 Amphifilidae H1 (env.FW) AB846664 Ciliophrys infusionum OTU 144 outgroup OTU 104

0.2 log10 read abundance 3 2 1 0 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. vesiculosus (UK) F . vesiculosus (NO) F . vesiculosus A . nodosum (NO) H . elonogata (UK) S . polyschides (UK) L . digitata (NO) L . digitata (UK) S . latissima (NO) serratus (UK) F . serratus (NO*) F . vesiculosus

EF024274 Elev 18S 660 (env.So) OTU 372 OTU 372 (99.3%) KY905096 Rhogostoma cylindrica (env.So) KY991047 Thecofilosea C34CL127 (env.FW) 0.94 OTU 347 OTU 347 (98.8%) DQ303924 Protaspis grandis AF411273 WHOI LI1 14 Thecofilosea EU545724 (env.S) OTU 281 (98.4%) OTU 281 0.78 0.85 KJ925270 271B10 (env.BW) OTU 199 (99.8) OTU 199 KX580625 Lecythium hyalinum 3 OTU 325 (95%) OTU 325 AY620333 7-1.3 (env.S) Novel Clade 3 OTU 83 (94.9%) OTU 83 KT818121 53c 43979 (env.W) Imbricatea OTU 215 (95.8%) OTU 215 KC243114 Thaumatomastix sp. (env.S/W)

0.95 KY991039 Glissomonadida C4CL114 (env.FW) OTU 259 (98.4%) Glissomonadida OTU 259 HM536169 Heteromita HFCC924 (env.So) AY884314 Neocercomonas 10-3.4 AY884327 Eocercomonas ramosa Cercomonadida GU385680 ME Euk FW80 (brown algae) OTU 54 (92.4%) OTU 54 OTU 124 (91.6%) OTU 124 OTU 110 (90.6%) OTU 110 0.94 OTU 86 (89.2%) OTU 86 AB622340 K9MAY2010 (env.FW) OTU 114 (86.6%) OTU 114 0.95 KC779514 Vampyrellida CAraX (env.S) Vampyrellida OTU 136 (85.2%) OTU 136 KC779515 Thalassomyxa MVa1x (env.S) 0.7 MF621963 Arachnomyxa cryptophaga AraCry01 (env.FW)

0.97 KP685352 F 13 2073 (env.S) 0.98 OTU 129 (93%) OTU 129 AB191413 TAGIRI5 (env.S) OTU 332 (95%) OTU 332 KX430467 Plasmodiophora brassicae

KX011114 Woronina pythii (oomycete) Phytomyxea EU567272 nh6 (marine) OTU 242 (92.6%) Novel Clade 9 AY268045 Sticholonche zanclea OTU 242 AB101542 Spongaster tetras outgroup AF063240 Acanthometra

0.1 log10 read abundance 2.5 2.0 1.5 1.0 0.5 0.0 0.88 0.07 0.43 bioRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.It 0.98 0.94 0.48 0.4 0.72 0.87 0.98 JX967274 AB971115 Env:RhizophydialesJp13Rp07E(env.FW) NG FJ804149 E15 (env.FW) AJ867631 Env:WS-10-E15 OTU AY032608 OTU on i AY364635 Nucleariapatters AF x2 AF OTU AY426915 Env:BL01032036(env.W) AB000960 KR336845 OTU 017174Rozellaallomycis OTU OTU OTU 349566 Nuclearia JN940944 372721 Env:BAQA128 (env.S) K XR_002431969.1 KU145487 OTU 364(95%) KU147486 OTU OTU 7 OTU JX096925 KM586996 F OTU 239(93.6%) doi: KX230692 OTU OTU 274(100%) AB971109 Env:Rhizophydiales(env.W) 330(99.5%) 122(98.9%) 349(96.2%) 036663 6 (99.8%) G EU192367 OTU OTU AF JQ OTU poridium daphniae Mitos U972082 Env:5H2cE8-16S(env.S) 181(99.8%) EU173355 Env:G OTU m PML2014- F Amoeboaphelidium 5 118(99.5%) https://doi.org/10.1101/2021.05.09.443287 AB053246 257(99.5%) ium penetrans Mesochytr 5 192(99.9%) 7 0 (99.5%) BAQA52 (env.S) 372708 Env:BAQA52 796369 DQ Chytridium polysiphoniae(brownalgae) KX017226 Nucleophaga OTU 7 (99.8%) 171(95%) 7 (99.3%) T 226(99.8%) Cystof 2 367(91.9%) aphrina DQ OTU 839595 eum (culture) Rhizophydium littor AY123299 5 (99.8%) wieringae(culture) Cryptococcus OTU onium Acrem albidus(culture) Cryptococcus Agaricomycot 322(98.3%) Cryptococcus Cryptococcus ladium Saroc 415278 ia Malassez 125(99.8%) iniatum (env.W) ilobasidium infirmom oebae saccam poridium Paramicros 225(100%) Issatchenkia 627144 HM plex sim ginica (typematerial) vir OTU (culture) Torulaspora delbueckii Urocystis colhici (culture) Urocystis made availableundera FR inia hordei(Poaceae) Pucc idium Melampsor 275(100%) E1T0 (env.A) 912P35RE1T0 856613 TVG-S004-0211 (env.S) F ina 33a05(env.A) LS06 (env.So) LS06 restricta (culture) R856610 AsA41 (aphid) AsA41 C FR scut andi C 856607 olae terric andi ulata (typematerial) da Candida oslonensis D01 D da deformans TDB1948 83-L (insect) CC-BY-NC-ND 4.0Internationallicense C andi ; outgroup this versionpostedMay10,2021. da galli Rozella Microsporidia & Aphelid

Chytrids Ascomycetes Basidiomycetes OTU 239 OTU 76 OTU 364 OTU 349 OTU 122 OTU 367 OTU 171 OTU 274 OTU 181 OTU 226 OTU 50 OTU 275 OTU 322 OTU 192 OTU 25 OTU 330 OTU 57 OTU 125 OTU 225 OTU 77 OTU 257 OTU 118 . The copyrightholderforthispreprint

F. serratus (UK) F. vesiculosus (NO*) F. vesiculosus (UK) F. vesiculosus (NO) Log10 readabundance A. nodosum (NO) H. elonogata (UK) 3 S. polyschides (UK) 2 L. digitata (NO) 1 L. digitata (UK)

0 S. latissima (NO) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A . nodosum (NO) serratus (UK) F . serratus (NO*) F . vesiculosus (UK) F . vesiculosus (NO) F . vesiculosus H . elonogata (UK) S . polyschides (UK) L . digitata (NO) L . digitata (UK) S . latissima (NO)

EF106789 Navicula glac iei CCMP2744 (culture) OTU 63 (98.3%) OTU 63 KY320365 Navicula salinarum (env.S) OTU 237 (99.8%) OTU 237 AF525663 Pseudogomphonema sp LM 2002 OTU 112 (98.3%) OTU 112 MH656614 Uncult. clone Acaer 35 C07 (GC copepod) OTU 234 (97.1%) OTU 234 OTU 68 (98.6%) OTU 68 KU867915 Navicula cf normaloides CU 2016a (env.Si) OTU 233 (99%) OTU 233 OTU 17 (99%) OTU 17 GU385626 ME Euk FW24 (brown algae, Ascophyllum nodosum) OTU 293 (90.5%) OTU 293 AM502030 Placoneis hambergii AT 160Gel09 (culture) AM502020 Navicula brockmannii strain AT 111Gel10 (culture) AB430614 Cocconeis stauroneiformis (culture) OTU 220 (98.8%) OTU 220 OTU 201 (95.9%) OTU 201 OTU 72 (97.6%) OTU 72 KY320345 Berkeleya rutilans strain TA440 (env.S) OTU 271 (100%) OTU 271 OTU 269 (99.8%) OTU 269 KY320352 Parlibellus delognei cf ellipticus strain TA387 (env.S) OTU 282 (97.3%) OTU 282 AM501960 Amphora pediculus strain AT 117 11 (culture) MH063494 Phaeodactylum tricornutum (culture) OTU 165 (95.5%) OTU 165 KR048195 Catacombas sp s0045 OTU 265 (99.8%) OTU 265 MG684378 Fragilariophyceae sp isolate Azo42a OTU 94 (99.8%) OTU 94 OTU 91 (99.8%) OTU 91 HQ912621 Synedra hyperborea strain CCMP1423 (culture) HQ912612 Licmophora paradoxa strain CCMP2313 (culture) OTU 205 (99.5%) OTU 205 AY633758 Licmophora gracilis (acoel, Convoluta convoluta) OTU 128 (99.5%) OTU 128 AB430607 Pteroncola inane strain s0247 (culture) OTU 93 (99.3%) OTU 93 JN975251 Rhabdonema arcuatum strain ECT3898 Rhabdo (culture) OTU 299 (98.3%) OTU 299 OTU 337 (99.8%) OTU 337 JX413544 Delphineis sp 1 MPA 2013 isolate ECT3886 delphineid OTU 172 (100%) OTU 172 JQ217341 Cyclotella choctawhatcheeana OTU 197 (99.5%) OTU 197 KY912618 Thalassiosira proschkinae strain SMDC305 (env.W) OTU 277 (98%) OTU 277 GU385575 ME Euk DBT67 (brown algae, Ascophyllum nodosum) FM877746 Diatom endosymbiont ex foraminifera (Neorotalia sp.) OTU 333 (100%) OTU 333 KY980247 Chaetoceros sp isolate BH46 158 OTU 101 (99.8%) OTU 101 KY852276 Chaetoceros socialis (env.W) OTU 317 (100%) OTU 317 DQ103771 Uncultured eukaryote clone M1 18B03 (env.S) KJ577861 Melosira sp TN 2014 isolate Bay Cut Melosira (culture) OTU 344 (86.4%) OTU 344 MN189660 Uncultured eukaryote (GC fish) OTU 162 (92.7%) KC309527 Corethron sp. (culture) OTU 162 JQ315679 Rhizosolenia delicatula strain KMMCC 1479 (culture) OTU 385 (99.5%) GBBA01013007T SA Saccharina latissima OTU 385 HQ710578 Fucus gardneri CNUK PF002 outgroup

0.06 Log10 read abundance 4 3 2 1 0 0.7 bioRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.It 1 0.67 0.7 0.78 x4 0.9 x4 0.86 0.99 0.66 Laminaria digitataclone 1 0.61 1 Saccharina latissimaclones AF KX827269 0.8 OTU OTU 1 KY987596 HE866895 HE866932 1 1 1 0.94 L43059 OTU OTU 3 123579 0.8 0.75 1 Laminaria digitataclones ECTOCARPUS SP. doi: CHORDA FILUM DQ AY232607 L43066 Ascophyllum nodosum 1 1 GQ MYRIOTRICHIA CLAVAEFORMIS 1 AB011423 0.73 MYRIONEMA STRANGULANS HQ KY233156 Ulvophycea e STREBLONEMA SP. OTU 146OTU MICROSPONGIUM TENUISIMUM KY987593 Fucus serratusclone OTU 31OTU ( Saccorhiza polyschides OTU 70OTU OTU OTU 2 Fucus serratusclone Fucus vesiculosusclone Fucus serratusclones 100% OTU 10OTU AY278216 781327 AD141 OTU OTU 5 OTU 41OTU OTU 326OTU HQ OTU OTU 9 0.8 1 Saccorhiza polyschides https://doi.org/10.1101/2021.05.09.443287 Fucus serratusclones Fucus serratusclones OTU 236OTU 433994 ( OTU 69OTU 710578 Fucus vesiculosusclones 99.5% Ecklonia cava Sacchari 1 Eisenia Desmarestia anceps OTU 105OTU Saccorhiza polyschidesclones Phaeurus antarcticus OTU 292OTU OTU 64OTU 0.39 129935 Scytosi OTU 149OTU OTU 49 49 OTU 1 1 0.99 Himanthalia elongata OTU 244OTU OTU OTU 4 OTU 348OTU OTU 113OTU ) OTU 121OTU OTU 23OTU OTU 381OTU 1 OTU 185 185 OTU AY303599 ( OTU 143OTU 1 Halothrix ambigua OTU 180OTU 99.8% ( ( 1 ( Fucus distichus ( 95.4% ( 94.7% ( OTU 26 26 OTU ) 98.6% 1 Silvetia sili Fucus gardner 99.5% 99.3% 99.0% ( Silvetia compressa OTU 59 59 OTU 88 OTU 40 OTU 96.6% Gomontia polyrhiza 1 ( OTU 340 340 OTU OTU 109 109 OTU ( na j 1 99.3% 96.8% ( OTU 99 99 OTU 35 OTU 123 OTU OTU 177 177 OTU 28 OTU phon lomentaria Pylaiella 98.8% ( OTU 377 377 OTU ( 1 92.5% ( ( 96.8% OTU 150 150 OTU 97.5% OTU 18OTU 97.2% aponica 1 ( AF OTU 7 OTU ) ( OTU 133OTU 97.5% ( OTU 43 43 OTU 97.2% ) ) OTU 216 216 OTU OTU 294 294 OTU ( ( 96.9% ) ( ( ) ) ) 96.8% OTU 346 346 OTU OTU 174 174 OTU OTU 368 368 OTU OTU 56 56 OTU 92.5% ( 91.7% 0.78 97.2% ) OTU 213 213 OTU 97.2% ( AB426255 ( 079788 ( ) AF OTU 389 389 OTU 93.2% Bolbocol ) ( 96.8% i 99.8% OTU 134OTU OTU OTU 6 ) 92.2% quosa ( ( ( made availableundera OTU 100 100 OTU MF JN ) 95.8% 91.5% 96.5% 0.98 OTU 87OTU ) AY303591 ) ) 499662 ( ( ( ( ( ) 95.4% 91.5% 95.4% ) 96.0% 91.7% OTU 127OTU OTU 51OTU ) ( 093105 ( ( ( ) ) 034614 94.0% 94.7% 95.1% clones ) ( 87.4% ) ) 95.4% ( ( ) ) ( ) 91.0% 94.2% Audouinella daviesii ( ) 99.8% ( ( 94.3% eon p clones ( ) ) ) 94.0% 91.7% ( ( ( 99.5% 93.0% 94.2% 94.5% ( Umbraulva j 94.5% ) ) ) Pseudendoclonium fucicola ( ( ) ) ( 94.1% clones 99.3% 99.8% ) ) ) ) Ulva linza (

) Lithotrichon pulchrum 94.0% ( Acrochaete leptoc haete iliferum 99.0% ) ) ( x2 ) 97.8% ) F. serratus F. serratus L26183 ) ) ) ) ) ) OTU OTU ) F. vesiculosus ) S. japonica,Ecklonia,Eisenia L. digitata ) A. nodosum ) ) S. polyschides ) ) aponica 21 OTU 264OTU Pylaiella, Halothrix, Scytosiphon Pylaiella, Halothrix, Streblonema, Ectocarpus Myriotrichia, Myrionema, Chorda, Microspongium, Brown endophytes: Ceramium rubrum H. elongata DQ OTU 229OTU AF ( OTU 108OTU Uncharacterised 99.5% HM560627 OTU 120OTU 022769 488402 CC-BY-NC-ND 4.0Internationallicense ( ? ) 90.4% , S.latissima ( ; 98.0% Ca Cryptopleura ramosa ( this versionpostedMay10,2021. 99.5% ( 99.8% Polysi llithamni ) ) ) phonia fucoides ) Green algae on coll abens

Red algae OTU 133 OTU 108 OTU 120 OTU 229 OTU 21 OTU 10 OTU 69 OTU 180 OTU 264 OTU 6 OTU 127 OTU 51 OTU 134 OTU 87 OTU 1 OTU 70 OTU 146 OTU 9 OTU 31 OTU 3 OTU 64 OTU 113 OTU 143 OTU 348 OTU 5 OTU 2 OTU 18 OTU 326 OTU 149 OTU 244 OTU 4 OTU 381 OTU 121 OTU 236 OTU 292 OTU 105 OTU 41 OTU 23 OTU 49 OTU 185 OTU 59 OTU 177 OTU 99 OTU 26 OTU 109 OTU 88 OTU 35 OTU 294 OTU 150 OTU 123 OTU 40 OTU 28 OTU 389 OTU 346 OTU 340 OTU 377 OTU 43 OTU 7 OTU 216 OTU 213 OTU 174 OTU 56 OTU 100 OTU 368 . The copyrightholderforthispreprint

F. serratus (UK)

F. vesiculosus (NO*)

F. vesiculosus (UK)

F. vesiculosus (NO)

A. nodosum (NO)

H. elonogata (UK) Log10 read abundance S. polyschides (UK) 6 L. digitata (NO) 4

2 L. digitata (UK)

0 S. latissima (NO) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

I II

III IV

V VI

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A. (With Phaeophyceae OTUs) B. (Without Phaeophyceae OTUs)

F. serratus (UK) A. nodosum (NO) 0.25 F. serratus (UK) 0.2 L. digitata (UK) Fucales F. vesiculosus (NO*) S. latissima (NO) S. latissima (NO) Laminariales S. polyschides (UK) Tilopteridales F. vesiculosus (NO*) F. vesiculosus (UK) S. polyschides (UK) 0.0 F. vesiculosus (UK) F. vesiculosus (NO) 0.00 F. vesiculosus (NO) L. digitata (UK)

A. nodosum (NO)

-0.2 NMDS2 NMDS2 L. digitata (NO)

-0.25 L. digitata (NO) -0.4

H. elonogata (UK) H. elonogata (UK) -0.6 -0.25 0.00 0.25 0.50 -0.25 0.00 0.25 NMDS1 NMDS1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.09.443287; this version posted May 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fucales Laminariales

125 (26) 20 (7) 31 (0)

25 (15) 22 (1) 3 (0)

39 (1)

Tilopteridales